REVIEW ARTICLE published: 11 November 2014 doi: 10.3389/fimmu.2014.00563 and cancer: the unintended consequences of anemia correction

Nataša Debeljak 1*, Peter Solár 2 and Arthur J. Sytkowski 3

1 Faculty of Medicine, Institute of Biochemistry, University of Ljubljana, Ljubljana, Slovenia 2 Department of Cell and Molecular Biology, Institute of Biology and Ecology, Faculty of Sciences, Pavol Jozef Šafárik University, Košice, Slovakia 3 Oncology Therapeutic Area, Quintiles Transnational, Arlington, MA, USA

Edited by: Until 1990, erythropoietin (EPO) was considered to have a single biological purpose and Pietro Ghezzi, Brighton and Sussex action, the stimulation of growth and differentiation. Slowly, scientific and Medical School, UK medical opinion evolved, beginning with the discovery of an effect on endothelial cell Reviewed by: Ralf Schindler, Charité, Germany growth in vitro and the identification of EPO receptors (EPORs) on neuronal cells. We Domenico Ribatti, University of Bari now know that EPO is a pleiotropic growth factor that exhibits an anti-apoptotic action on Medical School, Italy numerous cells and tissues, including malignant ones. In this article, we present a short Chris Thiemermann, Queen Mary discussion of EPO, receptors involved in EPO signal transduction, and their action on non- University of London, UK hematopoietic cells. This is followed by a more detailed presentation of both pre-clinical *Correspondence: Nataša Debeljak, Faculty of Medicine, and clinical data that demonstrate EPO’s action on cancer cells, as well as tumor angio- Institute of Biochemistry, University genesis and lymphangiogenesis. Clinical trials with reported adverse effects of chronic of Ljubljana, Vrazov trg 2, SI-1000 erythropoiesis-stimulating agents (ESAs) treatment as well as clinical studies exploring Ljubljana, Slovenia the prognostic significance of EPO and EPOR expression in cancer patients are reviewed. e-mail: [email protected] Finally, we address the use of EPO and other ESAs in cancer patients.

Keywords: erythropoietin, erythropoietin , receptor partners, cancer, cell response, , clinical trials

INTRODUCTION more detailed presentation of both pre-clinical and clinical data The presence of a circulating hemopoietic factor controlling the that demonstrate EPO’s diverse actions on cancer cells as well red blood cell (RBC) production was first suggested in 1906 (1). as possible receptors involved in the response of cancer cells to This humoral factor was experimentally confirmed almost half EPO/ESA therapy. Finally, we review current recommendations a decade later and the name erythropoietin was given (2). In for the use of rHuEPO and other ESAs as supportive therapy in 1977, the was isolated from human urine (3) enabling cancer patients with anemia that often develops during the radio- research toward cloning of the , its characterization, and or chemotherapy. in vitro expression (4, 5). Only 4 years later, the US Food and Drug Administration (FDA) approved the first commercially ERYTHROPOIETIN available recombinant human erythropoietin (rHuEPO), epoetin The human EPO gene spans over 3 kb and contains five exons alfa, for the treatment of anemia associated with chronic kid- encoding a 193 amino acid protein (4, 5). It is a single copy ney disease (CKD) (6). Later on, it was approved also for use gene located on 7 at position 7q22 (14, 15). A in patients with other anemias including cancer patients under- single splice variant of EPO gene is known (http://www.ncbi. going chemotherapy (7). Thereafter, rHuEPO became a leading nlm.nih.gov/gene/2056). is regulated by oxygen drug for treatment of anemia virtually abolishing the need for levels through hypoxia. Transcription factors involved are stim- RBC transfusion in some types of anemia. As a result, since ulatory HIF-2, HNF-4alpha and inhibitory GATA-2, NF-kappaB the 1990s, several new erythropoiesis-stimulating agents (ESA) [reviewed in Ref. (16, 17)]. have become available on the market or are under development During post-translation modification, the N-terminal 27 [reviewed in Ref. (8)]. amino acid signal is cleaved and R166 removed result- Erythropoietin (EPO) was first considered to have a single ing in a 165 amino acid mature protein (18). Urinary protein biological purpose and action – the stimulation of RBC growth containing 166 amino acids has also been characterized (19). and differentiation and, as such safe, for use in cancer patients. The single-chain protein is heavily glycosylated with a molecu- Slowly, scientific and medical opinion evolved, beginning with lar weight ranging from 30 to 39 kDa. Three N-linked (N24, N38, the discovery of an effect on endothelial cell growth in vitro (9) and N83) and one O-linked (S126) oligosaccharide side chains and the identification of EPO receptors (EPORs) on neuronal represent 35–40% of the EPO molecular mass. Protein structure is cells (10). We now know that EPO is a pleiotropic growth fac- stabilized with two intra-chain disulfide bridges between C7–C161 tor that exhibits an anti-apoptotic action on numerous cells and and C29–C33 (19, 20). N glycosylation does not affect hormone tissues, including malignant ones [reviewed in Ref. (11–13)]. In function in vitro but is essential for in vivo biological activity like this article, we present a short discussion of EPO, its signaling, biosynthesis, structural stability, secretion, plasma half-life, and and its action on non-hematopoietic cells. This is followed by a clearance (21–23).

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In adult human beings, the hormone is produced mainly by the EPO-EPOR signaling cascade. EPO has two non-identical binding renal cortex (24, 25), while in the developing fetus, the liver is the sites toward EPOR receptors, a high-affinity G151 (nano-molar) principal source (26). EPO is secreted into the bloodstream, circu- to the first, and a low-affinity R103 (micro-molar) interaction lates to the bone marrow, and binds to EPOR situated on the cell to the second receptor (33). Main signaling pathways activated surface of erythroid progenitors promoting their survival,prolifer- by EPO are JAK2/STAT5 pathway, phosphatidylinositol 3-kinase ation, and differentiation (27). EPO is also produced by numerous (PI3K) pathway, RAS/MAP kinase pathway, and protein kinase C non-hematopoietic cells and may act in endocrine, autocrine, and (PKC) pathway (34)(Figure 1). The JAK2/STAT5 and RAS/MAP paracrine manner (28). kinase (RAS-RAF-MEK-ERK) pathways are associated with hor- Commercially available rHuEPO has the same 165 amino acid mone mitogenic action, while the PI3K pathway (PI3K-AKT) is sequence as naturally occurring hormone (29). However, the level related with anti-apoptotic activities (27). of glycosylation in rHuEPO depends on the expression system used In non-hematopoietic tissue,some other receptor partners have (30). Glycosylation pattern can be analyzed by isoelectric focus- been proposed, including the beta common receptor (βcR) (35) ing enabling, thus distinguishing endogenous EPO (eEPO) from and the epinephrine B4 receptor (EPHB4) (36). The EPO mole- rHuEPO (31). Also, urinary and serum EPO have some minor cule was indicated to bind to the hetero-dimmer EPOR-EPHB4 heterogeneity in glycosylation levels (32). or hetero-trimmer EPOR-βcR-EPOR. Most probably, the high- affinity site is involved in binding to EPOR, while other receptor ERYTHROPOIETIN SIGNAL TRANSDUCTION partners are bound with low affinity or an alternative site result- In classical signal transduction in erythropoiesis, one EPO mole- ing in activation of different, tissue-protective part of EPO-EPOR cule binds to an EPOR homo-dimer leading to activation of the signaling cascade.

FIGURE 1 | Erythropoietin receptor and signaling pathways. The structure of the receptor dimer is outlined; docking sites for several intracellular are marked with P and linked with black-dotted arrow to individual pathway components. Positive interactions are presented with full black arrows, negative with dotted red.

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Erythropoietin receptor European patent application [EP 2 492 355 A1 (36)] dis- The human EPOR gene spans over 6 kb and contains 8 exons closes a molecular composition(s) of a novel tissue-protective encoding a 508 amino acid protein (37). The gene is located EPO-binding receptor protein complex, termed NEPOR. NEPOR on at position 19p13.3-p13.2 (38, 39). Sev- represents a novel EPOR derived from a unique combination of eral EPOR splice variants are known such as non-coding RNA, EPOR heteroreceptor dimer EPOR-EPHB4, homoreceptor EPOR- functional transmembrane protein (EPOR-F), truncated protein EPOR, and EPHB4-EPHB4, and other components derived from (EPOR-T), and at least one soluble variant (EPOR-S) (40–42) biology (Figure 3). (http://www.ncbi.nlm.nih.gov/gene/2057). EPO receptor is a member of the type I receptor super- Other partners family (43). During post-translation modification, the N-terminal Interaction of EPOR with several receptors has been indicated. 24-amino acid signal peptide is cleaved and the protein is modified Some interactions were suggested only on the level of receptor by glycosylation, phosphorylation, and ubiquitination to a mature 66–105 kDa protein (38, 44, 45). Mature human receptor consists of extracellular, single transmembrane, and cytoplasmic regions (46)(Figure 1). The majority of the EPOR is located on the cell surface of erythroid progenitors, erythroid burst-forming units (BFU-E), and erythroid colony-forming units (CFU-E) in the bone mar- row. However, the EPOR is expressed by various other tissues such as brain, heart, liver, and others where it is involved in the tissue protection. As EPOR is present in various cancer cells, the use of rHuEPO in cancer patients may be problematic due to potential activation of EPO-EPOR signaling pathways result- ing in tumor protection (anti-apoptotic action) or proliferation (mitogenic action).

Beta common receptor (βcR) The human colony-stimulating factor 2 receptor, beta (CSF2RB) gene contains 14 exons and is located on chromosome 22 at posi- tion 22q13.1 (http://www.ncbi.nlm.nih.gov/gene/1439). CSF2RB gene encodes β-common receptor (βcR), a common beta chain subunit of the high-affinity receptor for 3 (IL3), interleukin 5 (IL5), and CSF2 (granulocyte-macrophage colony- FIGURE 2 | EPOR-βcR receptor complex. Proposed structures of stimulating factor). tissue-protective complexes are presented: heterotrimer including βcR The interaction of EPOR with the βcR was discovered and homodimer with EPOR and heterodimer βcR with EPOR. Adapted from Brines and Cerami (47). tissue protection may be signaling through a heteroreceptor complex involving EPOR and βcR (35). This signaling net- work is still not understood but may involve signal pathways different from those triggered by EPOR-EPOR (47–50). The tissue-protective effects of EPO could be mediated by an EPOR heteroreceptor dimer EPOR-βcR or trimmer EPOR-βcR-EPOR (Figure 2).

Ephrin type-B receptor 4 (EPHB4) The human Ephrin receptor B4 (EPHB4) gene contains 17 exons and is located on chromosome 7 at position 7q22 (http://www. ncbi.nlm.nih.gov/gene/2050). Ephrin receptors were named Eph after the EPO-producing hepatocellular carcinoma cell line from which its cDNA was isolated. They form the largest family of (RTK) family. About 16 ephrin receptor (EphA1-10, EphB1-6) have been identified in the vertebrate genome (51), 14 of which are present in human beings. Ephrin receptors and their ligands, the , mediate numerus devel- opmental processes. The protein encoded by EPHB4 gene binds FIGURE 3 | EPOR-tissue-protective erythropoietin receptor (nepor to ephrin-B2 ligand and plays an essential role in vascular devel- receptor complex). Proposed structure of EPO interacting complexes are presented: homodimer EPHB4, homodimer EPOR, and heterodimer opment. EPHB4 was indicated also as survival factor in several EPHB4 with EPOR. Adapted from Jackson (36). cancers (52, 53).

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expression correlation (54, 55), others on adverse tumor response cells, where EPO-EPOR signaling is associated with increased cell to receptor-specific therapy (56, 57). The exact mechanisms of proliferation and/or survival, in tumor cells, the EPO-EPOR axis these correlations is not known yet but are being intensively does not always lead to increased proliferation but might increase studied. the resistance of cancer cells to different therapies. For more infor- Increased EPOR expression was related to a reduced response to mation about the role of EPO and its receptor in growth, survival, tamoxifen treatment, an estrogen receptor (ER) specific inhibitor and therapeutic response of human cancer cells, see the critical (56). Furthermore, EPOR receptor was shown to enhance ER review of Szenajch et al. (13), where all information based on activity and promote cell proliferation (58). Several correlations published EPO papers through 2010 is well summarized. between EPOR, ER, and other steroid receptors expression have Recently, the presence of EPOR signaling and EPO-induced been described (54, 55, 59, 60). cellular proliferation was confirmed in renal cancer cells (80, 82), The antagonistic effect of EPOR and human epidermal growth head and neck squamous cell carcinomas (77), and cervical cancer factor receptor-2 (HER2) co-expression on transtuzumab ther- cell lines (78), as well as glioma cells (87). apy, a HER2 inhibiting antibody has also been observed (57). The Erythropoietin-induced proliferation of cancer cells was associ- JAK2-mediated activation of SRC and inactivation of PTEN have ated with the activation of JAK2, JAK3, STAT3, and STAT5 but not been proposed as underlying mechanisms. JAK1 or STAT1 (78), AKT phosphorylation (77), ERK phospho- rylation (87) with hTERT gene transcription by JAK2/STAT5/c- ACTION OF EPO ON NON-HEMATOPOIETIC CELLS MYC, and hTERT protein phosphorylation by PI3K/AKT (88). EPO and its receptor have been identified in several non- Furthermore, the EPO-EPOR pathway stimulated the expression hematopoietic cells and tissue types like central nervous system, of cyclin D1 and inhibiting the expression of p21cip1 and p27kip1 heart, kidney, gastrointestinal system, reproductive tract, endothe- through the phosphorylation of JAK2 and ERK1/2, led to a more lium, and others [reviewed in Ref. (11, 12, 61)]. In these tissues, rapid progression through renal cancer cell cycle (82). Interest- EPO was shown to be tissue protective in an anti-apoptotic and/or ingly, EPO or (SCF) alone produced a modest mitogenic manner. Several ongoing pre-clinical and clinical trials number of cervical cancer cell colonies, whereas the combination are exploring the potential use of rHuEPO and other ESAs, such EPO/SCF induced a significantly more. Similarly, co-stimulation as tissue-protective agents in the brain, heart, and wound healing with EPO/SCF induced a significantly higher number of migrat- [reviewed in Ref. (62, 63)]. ing cervical cancer cells than either cytokine alone. Concurrently, Furthermore, EPORs have been found also on several types of EPO induced a modest, transient activation of ERK1/2, whereas tumors and malignant cells [reviewed in Ref. (64–67)] question- SCF and EPO/SCF prompted a strong, sustained phosphorylation ing the use of recombinant EPO in cancer patients (68). We will of ERK1/2 (89). now focus on a review of various effects of EPO and its receptor on Erythropoietin was also involved in cell growth, invasion, sur- cancer cells, being an update of EPO research reviewed by Szenajch vival, and sensitivity to the multikinase inhibitor and et al. (13). cisplatin in renal cancer cells (80) and in head and neck squamous cell carcinoma (77), respectively. In vitro, EPO had a protec- EPO AND CANCER CELLS RESPONSE tive effect on radiation-treated MDA-MB-435 cells; however, EPO The presence of functional EPOR was demonstrated in cancer treatment alone or combined with chemotherapy or hypoxia did stem cells (69–71). Different types of tumors as well as cell lines not influence cell survival. In vivo, EPO increased lung metas- have been found to express EPOR mRNA transcripts, which might tases in immunocompromised mice injected with MDA-MB-231 be translated into full-length EPOR as well as soluble or other or MDA-MB-435 cells and treated with chemotherapy relative to truncated forms (40). In this regard, Um et al. (72) measured inter- mice treated with chemotherapy alone (90). nalized 125I-EPO and found that just 50 high-affinity cell surface Very recently, Trošt et al. (55) confirmed the results of Arca- EPO binding sites were sufficient for EPO-mediated activation of soy et al. (91) with positive response of breast cancer cells to EPO. intracellular signal transduction in SH-SY5Y and PC-12 cancer Moreover, they demonstrated time- and concentration-dependent cells. EPOR expression has been demonstrated by flow cytometry manner of EPO-induced MCF-7 proliferation and EGR1, FOS, using a specific EPOR antibody in a panel of 29 tumor cell lines, and EPOR as transcription targets of the EPO-EPOR signal- including 18 adherent cell lines (73). ing loop (55). In this regard, Inbar et al. (92) discovered that Despite the fact that many tumor cells were confirmed to pos- EPO-driven EGR1 and c-FOS gene expression, as well as his- sess the EPOR, there is still some debate on stimulatory effects of tone H4 acetylation in breast cancer cells were mediated via EPO on these cells. On the one hand, there are papers pointing polyADP-ribosylation. EPO-induced breast cancer cell migration to the proliferative response of cancer cells after rHuEPO treat- was blocked by the PARP inhibitor Veliparib (ABT-888), suggest- ment (55, 74–82); on the other hand, some tumor cells, in spite of ing an essential role for polyADP-ribosylation in this process and evidence of EPOR functionality (67, 81), did not exhibit a growth suggesting a new cancer-associated anemia treatment modality response (67, 83–86). Our previous studies revealed a weak surface with combined administration of EPO and PARP inhibitors (92). EPOR signal in A2780 cells with most of EPOR found in the cyto- Recent research revealed that EPO/EPOR contributes to the plasm, more abundantly as an intracellular membrane-associated mechanism of resistance in breast cancer cell line protein than a soluble one. Silencing EPOR expression resulted SKBR3, and EPOR downregulation can reverse the resistance in reduced A2780 proliferation as well as a reduction in EPO- to trastuzumab. EPOR expression may be involved in tumor induced phosphorylation of ERK1/2 (81). Unlike hematopoietic progression and proliferation in HER2-positive breast cancer

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(93). Indeed, EPO antagonized trastuzumab-induced therapeutic phosphorylation of STAT-5 in HUVEC cells, but only very weakly effects through JAK2-mediated activation of SRC and inactiva- in smooth muscle cells (100). Results of our group revealed that tion of PTEN protein, so combined therapy of HER2-positive conditioned media of EPO treated A2780 cells under hypoxic con- cancer cells with EPO and trastuzumab reduced the response ditions induced significant STAT-5 phosphorylation, as well as of these cells to trastuzumab both in vitro and in vivo. Further- proliferation of HUVEC cells. A new finding is the fact that pro- more, concurrent administration of EPO and trastuzumab corre- stimulatory effect of hypoxic A2780 media was partly mediated lated with shorter progression-free and overall survival in patients by EPO. Furthermore, EPO increased secretion of IL-4, IL-5, IL-6, with HER2-positive metastatic breast cancer (57). The mecha- IL-8, IL-10, IL-12, IL-13, GM-CSF, and IFN γ by A2780 cells in nism of EPO-EPOR and HER2 co-regulation in breast cancer hypoxic conditions (101). was confirmed by miR-125b, which is downregulated in metasta- An in vivo angiogenic potential of EPO was originally demon- tic breast cancers and a significant positive correlation between strated by Yasuda et al. (102) who found that injection of EPO EPOR and HER2 levels that are both targets of miR-125b was into the ovariectomized mouse uterine cavity promoted blood demonstrated (94). vessel formation of the endometrium. Similarly, Ribatti et al. Because of adverse tumor response and/or poorer survival in (103) demonstrated that EPO induced a potent in vivo angio- ESA-treated cancer patients, studies of EPO effects on cancer stem genic response of the chick embryo chorioallantoic membrane. cells was initiated. Cao et al. (69) found that glioma stem cells Furthermore, the role of EPO in physiological angiogenesis was (GSC) express higher levels of EPOR than matched non-stem described during wound healing and in the developing of mouse glioma cells. They targeted EPOR expression in GSG with shRNA embryo (104, 105). and reduced growth, survival, and neurosphere formation capac- The study of Yasuda et al. (106) revealed that normal human ity, so confirmed the role for EPOR in GSC maintenance. A small cervix and endometrium, as well as ovary malignant tumors of molecule inhibitor of STAT3 led to reduced GSG growth and sur- female reproductive organs produce EPO and EPOR, and that vival. EPO-EPOR signaling was also critical for survival in vivo, as the tumor cells themselves and capillary endothelial cells are sites targeting EPOR expression decreased GSC tumorigenic potential responsive to the EPO signal. Yasuda et al. (107) proposed the (69). Furthermore, Todaro et al. (71) showed that breast cancer presence of a paracrine or autocrine EPO-EPOR loop and its con- stem-like cells (BCSC) isolated from patient tumors express the tribution to tumorigenesis in female reproductive organs based EPOR and respond to EPO treatment with increased proliferation on the mitogenic action of EPO as well as on the finding that and self-renewal. Importantly, EPO stimulation increased BCSC injection of soluble EPOR (EPOR-S) or EPO-monoclonal anti- survival and resistance to chemotherapeutic agents, probably by body into blocks of tumor specimens was followed by EPO-activated AKT and ERK pathways and promoted metasta- of tumor cells and endothelial cells. Although some studies have tic progression of tumor xenografts in the presence and in the not confirmed a direct stimulatory effect of EPO on tumor cells, absence of chemotherapy treatment. These results suggest that there is ample evidence of this effect on endothelial cell prolif- EPO acts directly on BCSC by activating specific survival pathways, eration and/or angiogenesis of tumors. In this regard, EPO xin- resulting in BCSC protection from chemotherapy and enhanced duced angiogenesis in chemically induced murine hepatic tumors tumor progression (71). Moreover, EPO/EPOR promoted tumori- (108) and accelerated the growth of EPOR negative Lewis lung genesis in genetically engineered mouse models of breast cancer carcinoma cells by promoting tumor angiogenesis in vivo (109). by activating JAK/STAT signaling in breast tumor-initiating cells Interestingly, an EPO analog stimulated neovascularization (TICs) and promoted its self-renewal. EPO gene expression cor- in colorectal liver metastases of hepatectomized and non- related with shortened relapse-free survival and pharmacologic hepatectomized mice (110). Moreover, Nico et al. (111) demon- JAK2 inhibition revealed a synergistic effect with chemotherapy in strated that EPO secreted by glioma tumor cells affected glioma tumor growth inhibition in vivo (70). vascular endothelial cells and promoted angiogenesis in a paracrine manner. Specificity of the EPO effect was shown through EPO AND TUMOR ANGIOGENESIS AND LYMPHANGIOGENESIS an anti-EPO antibody, which was able to significantly inhibit the In 1990, Anagnostou et al. (9) found that EPO enhances the pro- angiogenesis response. Despite the absence of melanoma growth liferation and migration of human umbilical vein endothelial cells stimulation in vivo, EPO increased vascular size in the xenografts. and bovine adrenal capillary endothelial cells (95, 96) and demon- Indeed, EPO-induced angiogenesis in Matrigel plug assays, and strated the presence of EPOR mRNA in human umbilical vein neutralization of EPO secreted by melanoma cells resulted in endothelial cells, as well as strong positive EPOR protein staining decreased angiogenesis, which supports the role of EPO/EPOR in of the vascular endothelium in vivo (97). The presence of EPOR melanoma progression via angiogenesis stimulation (112). Even was also shown by Yamajiet al. (98), who suggested that brain cap- more interestingly, EPO accelerated the tumor growth of MMQ illary endothelial cells express not only an authentic form of EPOR pituitary adenoma xenografts lacking EPOR via enhancement of (EPOR-F) but also a soluble one (EPOR-S) and that EPO acts angiogenesis in vivo, without a direct EPO effect on MMQ cells directly on brain capillary endothelial cells as a competence factor. in vitro. EPO administration increased phosphorylation of JAK2, EPO signaling as a mitogen of endothelial cells was conducted via STAT3, and VEGF expression in HUVEC cells in vitro and in tyrosine phosphorylation of proteins including phosphorylation MMQ cell xenografts in vivo (113). The authors suggest that EPO of transcription factor STAT-5, which is similar to that occur- administration may promote the growth of pituitary adenomas by ring in erythroid cells (99). Moreover, experiments performed in enhancing angiogenesis through EPO-JAK2-STAT3-VEGF signal- cultured vascular cells demonstrated that EPO robustly induced ing pathway and should be used with caution in anemia patients

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Table 1 | Clinical trials with reported effects of ESA in cancer patients performed between 2009 and 2014.

Study (reference) Type No. of patients Therapy Support Hb control Disease control Observations (cancer type) therapy

Mäenpää et al. (138) N = 114 (various) EB ↑HRQoL

Thomaidis et al. (139) Ph2 N = 118 (esophagogastric) Cx EB ↑Hb (ESA) ↑OS, RFS (ESA)

AGO-ETC Moebus et al. N = 1.284 (breast) Cx EA ↑Hb, ↓TF (ESA) ↔OS, RFS ↑TE (ESA) (123)

GINECO Weber et al. Ph2 N = 100 (ovarian) Cx EA ↓NT (140)

Ohashi et al. (141) Meta N = 511 (CIA, various) Cx DP,EB ↓TF (ESA) ↔OS, RFS ↔TE

Michallet et al. (142) N = 107 (leukemia) Cx, T ESA ↑Hb, ↓TF (ESA) ↔OS, RFS ↑HRQoL (ESA), ↔TE

Tonia et al. (124) Meta N = 20.102, 91 trials ESA ↓TF (ESA) ↑TE, HRQoL (ESA)

Stehman et al. (143) N = 1.864 (ovarian) Cx G-CSF,ESA ↔OS multivariate analysis

CHOICE Aerts et al. N = 1.887 (various) Cx DP ↑Hb (ESA) Five severe drug (144); Van Belle et al. reactions (145, 146)

Kerkhofs et al. (147) N = 113 Cx EA ↑Hb (ESA) NR

Canon et al. (125) Ph3 N = 705 Cx DP ↑Hb (ESA) ↑TE (ESA)

Bustos et al. (126) N = 685 (CIA, various) Cx DP ↑Hb (ESA) ↑TE (ESA)

Cabanillas et al. (148)N = 109 (leukemia, Cx EA ↓TF (ESA) ↔CR ↔HRQoL lymphoma)

Cantrell et al. (149) N = 343 (CIA, ovarian) Cx ESA ↔OS, PFS

Fujisaka et al. (127) Ph3 N = 186 (CIA, various) Cx EB ↑Hb (ESA) ↔OS ↑HRQoL, ↑TE (ESA)

NOGGO-AGO Blohmer Ph3 N = 257 (cervical) Cx, Rx EA ↑Hb (ESA) ↑RFS (ESA), ↔OS ↔TE et al. (150)

PREPARE Untch et al. Ph3 N = 733 (breast) Cx, Na DP ↑Hb (ESA) ↓RFS (ESA) ↑TE (ESA) (128, 129)

Nagel et al. (151) Ph2 N = 74 (SCLC) Cx DP ↓TF (ESA) ↔PFS

RETRA Eisterer et al. N = 309 (various) Cx DP ↓TF (ESA) (152)

Djavan et al. (153) N = 1.567 (prostate) S ESA ↔RFS

Villegas et al. (130) Ph2 N = 44 (MDS) DP ↑TE (ESA)

Rørth et al. (154)N = 16 (solid) Cx DP ↑Hb (ESA) ↑HRQoL (ESA)

Tjulandin et al. (155) N = 186 (CIA, various) Cx ET ↑Hb, ↓TF (ESA) ↔AE

Esquerdo et al. (156) N = 100 (CIA, solid) Cx DP ↑Hb, ↓TF (ESA)

Chavez-MacGregor Retro N = 2.266 (breast) Cx ESA ↑TE (ESA) et al. (131)

Gómez et al. (157) (gastrointestinal) Cx EB ↑Hb (ESA) NR

Gomez-Alamillo et al. N = 22 (solid) Rx, Na EB ↑Hb (ESA) (158)

(Continued)

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Table 1 | Continued

Study (reference) Type No. of patients Therapy Support Hb control Disease control Observations (cancer type) therapy

Schwartzberg et al. Ph2 N = 752 (CIA, various) Cx DP ↑Hb (ESA) ↔AE (159)

Ray-Coquard et al. (160) N = 2.912 (solid) Cx DP ↑Hb (ESA)

Pronzato et al. (161) N = 223 (breast) Cx EA ↑Hb (ESA) ↔OS, TR, AE ↑PRO, HRQoL (ESA)

Vargas et al. (162) Ph4 N = 157 (CIA, various) Cx, Rx EA ↑Hb, ↓TF (ESA) ↑AE (ESA)

Auerbach et al. (163) Ph2 N = 242 (solid) Cx, DP,Fe ↑Hb (Fe) ↔AE

Maccio et al. (164) N = 148 (various) Cx EB, Fe ↔Hb (Fe)

Ichinose et al. (165) Ph2 N = 132 (lung, ovarian) Cx DP ↑Hb (ESA) ↑HRQoL (ESA)

GHSG HD15EPO N = 1.379 (HL) Cx EA ↓TF (ESA) ↔OS, RFS, PFS, ↔TE Engert et al. (166)

Gascon et al. (135) Ph2 N = 153 (NSCLC) Cx CERA, DP ↔Hb ↓OS (ESA) Terminated early

Muravyov et al. (167) N = 40 (solid) Cx EB ↑Hb (ESA)

Stull et al. (168) N = 1.494 (various) Cx DP ↑Hb (ESA) ↑HRQoL (ESA)

Tjulandin et al. (169) N = 223 (CIA, various) Cx ET, EA ↑Hb (ESA)

Roddy et al. (132) N = 79 (colorectal) Cx ESA ↑TE (ESA)

Hoskin et al. (170) N = 301 (head and neck) Rx EA ↑Hb (ESA) ↔OS, RFS ↔AE

Vansteenkiste et al. Ph3 N = 705 (CIA, solid) Cx DP ↑Hb, ↓TF (ESA) (171)

Grobmyer et al. (172) N = 40 (abdominal) S EA ↔TF

BRAVE Aapro et al. N = 463 (breast) Cx EB, At ↑TE (ESA), ↓TE (ESA, (133) At)

Aapro et al. (134) Meta N = 2.297 (breast) Cx EB ↔OS ↑TE (ESA),

Hernandez et al. (173) Ph3 N = 386 (CIA, solid) Cx DP ↓TF (ESA) ↔HRQoL, TE,

Greenberg et al. (174) Ph3 N = 110 (MDS) Cx ESA, GCSF ↔OS ↑HRQoL (ESA)

Repetto and CIPOMO N = 1.175 (various) Cx ESA, GCSF Investigators (175)

Bohlius et al. (136) Meta N = 13.933 (various) ESA ↓OS (over ↑Hb)

Lambin et al. (137) N = 1.397 (head and neck) Cx, Rx ESA ↓OS (over ↑Hb)

Tzekova et al. (176) Ph3 N = 216 (solid) Cx EZ ↑Hb (ESA) ↓TE (EZ), ↑HRQoL (ESA)

Study type: Meta, meta-analysis; Ph2-4, clinical study phase 2–4; Retro, retro-analysis. Disease/cancer type: CIA, chemotherapy-induced anemia; HL, Hodgkin’s lymphoma; MDS, myelodysplastic syndrome; NSCLC, non-small-cell lung cancer; SCLC, small-cell lung cancer. Therapy: At, antithrombotic; CERA, continuous ery- thropoietin receptor activator; Cx, chemotherapy; DP, darbepoetin; EA, epoetin alpha; EB, ; ESA, erythropoiesis-stimulating agent; ET, ; EZ, epoetin zeta; Fe, iron supplementation; Na, neoadjuvant; Rx, radiotherapy; S, surgery; TF, transfusion; T, transplantation. Disease control and observations: AF, adverse effects; CR, complete remission; DFS, disease-free survival; Hb, hemoglobin; HRQoL, health-related quality of life; NR, not reported; NT, neurotoxicity; OS, overall survival; PFS, progression-free survival; PRO, patient-reported outcomes; RFS, relapse-free survival; TE, thrombotic events; TR, tumor response; ↑/↓/↔, increased/decreased/no significant difference. Studies with indicated negative effects are marked in gray.

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Table 2 | Clinical studies exploring prognostic significance of EPO and EPOR expression in cancer patients.

Study (reference) No. of patients Therapy EPO expression EPOR expression Other observations (cancer type) as PF (method) as PF (method)

Seibold et al. (178) N = 114 (head and S, Rx, IPF – no EPO: ↑LRC, IPF – no EPOR: ↑OS neck SCC) ↑MFS, ↑OS

Lin et al. (180) N = 256 (oral SCC) S / IPF – ↑EPOR: ↑ATB, ↓OS, ↓DSS (qPCR, WB, IHC)

Wang et al. (120) N = 172 (GAC) IPF – ↓OS; ↑EPO: ↑EPOR, ↑EPOR: ↑ATB, ↑MD (IHC) ↑VEGF-↓EPOR ↑ATB, ↑MD (IHC)

Welsch et al. (177) N = 104 (PDAC) IPF, ↑sEPO: ↓OS (qPCR, (qPCR, IHC) ELISA, IHC)

Gombos et al. (184) N = 24 (colorectal AC) ↑EPO (IHC, qPCR, WB) ↑EPOR (IHC, qPCR, WB) ↑EPO and EPOR in ischemia/necrosis

Beschorner et al. (185) N = 43 (CPT) / ↓EPOR (IHC, qPCR, WB)

Rades et al. (179) N = 62 (NSCLC) Rx IPF; no EPO: ↑LRC, ↑OS ↑EPO and ↑EPOR: ↓PF

Volgger et al. (181) N = 107 (breast) ESA / ↑EPOR: ↑ER and ↑PR, ↑CRR, ↔OS (IHC, qPCR, WB)

Xu et al. (186) N = 96 (prostate: PCa, ↑EPO (BPH) (IHC) ↑EPOR (PCa, PIN) (IHC) PIN, BPH)

Liang et al. (57) N = 55/37 (breast) TZ, ESA / ↑EPOR and ↑HER2: ↓TR to TZ, ↓PFS, ↓OS (IHC)

Mirmoham-medsadegh N = 20 (melanoma) / ↑EPOR (qPCR, IHC, WB) et al. (187)

Giatromanolaki et al. N = 72 (endometry) / ↑EPOR: ↑ATB, ↓PF (IHC) ↑EPOR-↑HIF1α- (182) ↑VEGF

Larsson et al. (56) N = 500 (breast: TAM (qPCR, ELISA) ↑EPOR: ↓TR to TAM (qPCR, ER + , PR +) IHC)

Li et al. (119) N = 65 (tongue SCC) S IPF (IHC) IPF (IHC)

Miller et al. (73) N = 159 (various) ESA (qPCR) ↔PFS, ↑EPOR: ↓PFS in JAK2, HSP70 unresected T (qPCR)

Küster et al. (183) N = 131 (meningioma) / ↓EPOR: ↑CRR (IHC, qPCR, EPOR-F,EPOR-T, WB) EPOR-S

Disease/cancer type: BPH, benign prostatic hyperplasia; CPT, choroid plexus tumors (glioma, meningioma); ER+, estrogen receptor positive; GAC, gastric adenocar- cinoma; NSCLC, non-small-cell lung cancer; PR+, progesterone receptor positive; SCC, squamous cell carcinoma; PCa, prostate carcinoma; PDAC, pancreatic ductal adenocarcinoma; PIN, prostate intraepithelial neoplasia.Therapy: ESA, erythropoiesis-stimulating agent; Rx, radiotherapy; S, surgery;TAM, tamoxifen;TZ, trastuzumab. Prognostic factor and observations: ATB, aggressive tumor behavior (TNM stage, T and N classification); CRR, cancer recurrence rate; DSS, disease-specific survival; IPF, independent prognostic factor; LRC, loco-regional control; MD, microvessel density; MFS, metastases-free survival; OS, overall survival; PF, prognostic factor; PFS, progression-free survival; sEPO, serum EPO;TR, tumor response; ↑/↓/↔, increased/decreased/no significant difference. Methods: IHC, immunohistochemistry; qPCR, quantitative PCR; WB, Western blotting. bearing pituitary adenoma due to its potential deleterious effects EPO/EPOR levels correlated well with angiogenesis and progres- (113). On the contrary, very recently Pascual et al. (114) found sion of patients with hepatocellular carcinoma, neuroblastoma, that preoperative administration of EPO stimulates tumor recur- squamous cell carcinoma of the tongue, melanoma, and gastric rence in an animal model of colon cancer, but no evidence of adenocarcinoma (115–120). increased angiogenesis or enhanced-cell proliferation as possible The lymph node as a new target of EPO was presented by Lee mechanisms of EPO-induced recurrence was seen. Importantly, et al. (121) They showed that EPO can stimulate both lymph node

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lymphangiogenesis and nodal metastasis by increased migration, Based on discussed clinical studies, the adverse effect of capillary-like tube formation, and dose- and time-dependent pro- EPO/ESA in cancer patients during anti-cancer therapy could be liferation of human lymphatic endothelial cells in tumor-bearing related to chronic EPO treatment. Such EPO/ESA treatments are animals. Intraperitoneal administration of EPO induced AKT and long termed and rarely single dosed. We suggest that the con- ERK1/2 signalization followed by peritoneal lymphangiogenesis current use of long-termed EPO/ESA and anti-cancer treatment stimulation. Furthermore, systemic treatment of EPO increased is the main reason for EPO/ESA negative effects on response to infiltration of CD11b(+) macrophages in tumor-draining lymph anti-cancer therapy, overall survival, and disease recurrence. The nodes and increased VEGF-C expression in lymph node-derived mechanisms are still not well understood and more profound mol- CD11b(+) macrophages, as well as in bone marrow-derived ecular and biochemical characterization is needed; however, they macrophages in a dose- and time-dependent manner (121). may be linked to one of the EPO/ESA mechanisms indicated in the According to McKinney and Arcasoy (122),there are two poten- previous chapter. EPOR, βcR, and EPHB4 were previously shown tial mechanisms by which rHuEPO therapy may promote tumor to be expressed in various tumors. We do speculate that one of progression and reduce survival in some cancer patients: (1) these receptors and/or possible analogs of these receptors may be rHuEPO therapy may exert local effects in tumors, acting directly involved in the response of cancer cells to EPO/ESA therapy. on tumor cells or other cell types in the tumor microenviron- ment, such as the vascular endothelium and tumor-associated CONCLUSION macrophages or (2) rHuEPO may cause systemic effects that indi- A plethora of scientific evidence demonstrates a growth- rectly alter tumor biology in an unfavorable manner or directly promoting, anti-apoptotic action of EPO and other ESAs on give rise to specific systemic toxicities that impair survival. In non-hematopoietic cells, both normal and malignant, and this this regard, elevated hemoglobin, increased viscosity, platelet acti- is supported by numerous clinical observations showing adverse vation, endothelial progenitor mobilization, immunomodulatory effects of EPO administration on the clinical management of effects, and others could play significant roles. We add a third tumor growth and progression. As anticipated just a few years ago potential mechanism and this is direct effect of EPO on cancer stem (28),physicians who care for anemic cancer patients have been fac- and/or TICs, which could explain enhanced tumor progression ing a dilemma, whether to treat the anemic patient with an ESA, and poor survival of some cancer patients treated with EPO. thereby potentially increasing the risk of worsening the malig- nancy,or to withhold ESA treatment,with resultant patient fatigue, EPO (ESA) AND CLINICAL STUDIES reduced physical activity, increased hypoxic stress, and reliance on Several clinical trials have addressed the effects of one of the transfusion therapy. Primary tumors are not yet EPOR typed (like EPO/ESA treatments on disease control in cancer patients on co- breast cancers are assessed for ER/PR expression) though this idea temporal anti-cancer therapy,such as chemotherapy,radiotherapy, should be considered. There has been much discussion of EPO use neoadjuvant therapy, and surgery. Such EPO/ESA treatments are in cancer patients, and several professional and regulatory orga- long termed and rarely single dosed. The review of clinical trials nizations and authorities have issued various guidances. Perhaps, performed between 2009 and 2014 is presented in Table 1,being an the following “rule” used by several clinicians interviewed by one update of review by Szenajch et al. (13). Some of the studies indi- of the authors should be considered. If the cancer patient is being cate a negative outcome (marked in gray). Most of the reported treated with curative intent, avoid the use of ESAs. If the treatment negative effects are due to increased thrombotic events,a complica- plan is more conservative or palliative, consider ESAs for anemia tion driven by an increased number of RBC (123–134). However, treatment, but proceed with great caution. some of the studies also indicate a reduced recurrence free sur- vival and overall survival (128, 129, 135–137). Negative effects on AUTHOR CONTRIBUTIONS overall survival were already shown previously by several studies, Nataša Debeljak outlined the work. All authors designed and reviewed by Szenajch et al. (13). drafted the work. Arthur J. Sytkowski critically revised the work. Clinical studies exploring the prognostic significance of EPO All authors approved final version of the work and agreed to be and EPOR expression in cancer patients have also been explored accountable for all aspects of the work. and are listed in Table 2. Several studies indicated increased EPO expression as prognostic factor for reduced overall survival ACKNOWLEDGMENTS (120, 177) or vice versa, no EPO expression as prognostic factor Nataša Debeljak was supported by the J3-0124 grant to ND and P1- for increased overall survival (178, 179). Furthermore, increased 0104 to Radovan Komel,both from the Slovenian ResearchAgency. EPOR expression was identified as prognostic factor for reduced Peter Solár was partly supported by Scientific Grant Agency of the overall survival, more aggressive tumor behavior, and progression- Ministry of Education of the Slovak Republic under contract no. free survival (57, 73, 120, 180–182). Increased EPOR expression VEGA 1/0733/12. Arthur J. Sytkowski acknowledges the support of was also linked to reduced response to anti-cancer treatment (56, Quintiles Transnational where he is an executive director,hematol- 57). While no or reduced EPOR expression was identified as prog- ogy and oncology, in the Therapeutic Delivery Unit. The authors nostic factor for increased overall survival (178) and in contrast, thank Klemen Španinger for contribution of Figure 1. also to increased cancer recurrence rate (183). 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Effect as a potential conflict of interest. of epoetin alfa on survival and cancer treatment-related anemia and fatigue in patients receiving radical radiotherapy with curative intent for head and neck Received: 31 July 2014; accepted: 22 October 2014; published online: 11 November cancer. J Clin Oncol (2009) 27(34):5751–6. doi:10.1200/JCO.2009.22.3693 2014. 171. Vansteenkiste J, Dooms C, De Leyn P. The multidisciplinarity of stage III non- Citation: Debeljak N, Solár P and Sytkowski AJ (2014) Erythropoietin and can- small cell lung cancer. Eur J Cancer (2009) 45(Suppl 1):92–105. doi:10.1016/ cer: the unintended consequences of anemia correction. Front. Immunol. 5:563. doi: S0959-8049(09)70021-8 10.3389/fimmu.2014.00563 172. Grobmyer SR, Hemming AW, Harris N, Behrns K, Logan H, Kim RD, et al. This article was submitted to Inflammation, a section of the journal Frontiers in A pilot prospective randomized trial of postoperative epoetin alfa in patients Immunology. undergoing major operation for upper gastrointestinal malignancy. Am J Clin Copyright © 2014 Debeljak, Solár and Sytkowski. This is an open-access article dis- Oncol (2009) 32(6):570–3. doi:10.1097/COC.0b013e31819790a8 tributed under the terms of the Creative Commons Attribution License (CC BY). The 173. Hernandez E, Ganly P, Charu V, Dibenedetto J, Tomita D, Lillie T, et al. Ran- use, distribution or reproduction in other forums is permitted, provided the original domized, double-blind, placebo-controlled trial of every-3-week darbepoetin author(s) or licensor are credited and that the original publication in this journal is cited, alfa 300 micrograms for treatment of chemotherapy-induced anemia. Curr in accordance with accepted academic practice. No use, distribution or reproduction is Med Res Opin (2009) 25(9):2109–20. doi:10.1185/03007990903084164 permitted which does not comply with these terms.

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